3. Result and Discussion
3.1 Structural determination of compounds isolated from A. scoparia- 28
3.3.4 Effect of crude extract and its solvent fractions on cell migration
MMPs, especially MMP-2, are known to be involved in cell migration and invasion (Okoli, 2014). Therefore, The patterns of cell migration affected by sample treatment were examined through wound healing assay. Cells not treated with the sample (control) showed an obvious migration after 24 hours, while the cells treated with it did that migration was suppressed. Among samples tested, the water fraction little exhibited the inhibitory effect on cell migration. However, crude extract, n-hexane, 85% aq.MeOH and n-BuOH fractions were effective in inhibiting cell migration (Fig. 14).
Fig. 14. Effect of crude extract and its solvent fractions from A. scoparia on migration ability in HT-1080 cells.ㄹ ㄹㄹㄹ
3.3.5 Effect of crude extract and its solvent fractions from A. scoparia on mRNA levels of MMP-9 and 2 by RT-PCR
RT-PCR was performed to confirm mRNA expression levels of MMP-9 and MMP-2 in PMA-stimulated HT-1080 cells treated with each of crude extract and its solvent fractions of A. scoparia. β-Actin was used as the internal control.
In cells treated with PMA alone, mRNA expression of MMP-9 and MMP-2 increased compared to cells not treated with both PMA and samples. The crude extract reduced mRNA expression levels of MMP-9 and MMP-2 to 54.9%
and 52.5%, respectively (Fig. 15a).
In case of solvent fractions treatment, mRNA expression levels of MMP-9 and MMP-2 decreased except water fraction. n-Hexane fraction suppressed mRNA expression levels of MMP-9 and MMP-2 by 57.7% and 63.7%, showing the highest inhibitory effect, respectively. In addition, the 85% aq.MeOH fraction reduced the MMP-9 and MMP-2 mRNA expression levels to 70% and 74.1%, respectively and the n-BuOH fraction also reduced the MMP-9 and MMP-2 mRNA expression levels to 79% and 68.5%, respectively (Fig. 15b).
Fig. 15. Effect of crude extract (a) and its solvent fractions (b) of A. scoparia o n mRN A l e ve l s o f MMP - 9 and 2 d e t e r m i n e d b y R T - P C R . β-Actin was used as the internal standard. Band sizes were calculated and depicted as percentage compared to control group.
Values were normalized against house keeping β-actin mRNA levels.
V a l u e s a r e m e a n s ± S D . a - eM e a n s w i t h d i f f e r e n t letters at the same concentration are significantly different (p<0.05) by Duncan’s multiple range test.ddddddddddddddddddddddddd
3.3.6 Effect of crude extract and its solvent fractions on protein levels of MMP-9 and 2 by Western blot assay
Western blot assay was performed to confirm the protein expression levels of MMP-9 and MMP-2 in PMA-stimulated HT-1080 cells treated with each of crude extracts and its solvent fractions. In cells treated with PMA alone, protein expression of MMP-9 and MMP-2 increased compared to the blank.
The crude extract reduced protein expression levels of MMP-9 and MMP-2 to 35.4% and 23.9% in PMA-stimulated HT-1080 cells, compared to the control, respectively (Fig. 16a).
Protein expression levels of MMP-9 and MMP-2 also decreased by treatment of the solvent fractions. Especially, n-hexane and 85% aq.MeOH fractions were most effective in reducing protein expression of MMP-9 and MMP-2:
n-hexane fraction, 43.4% for MMP-9, 60.2% for MMP-2; 85% aq.MeOH fraction, 42.2% for MMP-9, 60.2% for MMP-2 (Fig. 16b).
Fig. 16. Effect of crude extract (a) and its solvent fractions (b) from A.
scoparia on protein levels of MMP-9 and 2 determined by Western blot assay. β-Actin was used as an internal standard. Band sizes were calculated and depicted as percentage compared to control group. Values were normalized against house keeping β-actin protein levels. Values are means ± SD. a-eMeans with different letters at the same concentration are significantly different (p<0.05) by Duncan’s multiple range test. ddddddddddddddddddddddd
4. Conclusion
Obesity-related diseases and cancers are on the rise worldwide, and The resulting mortality rate in Korea is a high proportion of all deaths. Obesity can be caused by several factors - mental tension, lack of physical exercise, irregular eating habits inescapable in modern society, etc. In addition, chronic inflammation due to excessive accumulation of fat can affect organs closely related with cancer, eventually leading into cancer. These cancer or tumor cells also metastasize to other organs, which can be fatal to patients. Matrix metalloproteinases, enzymes involved in invasion, angiogenesis, and metastasis of cancer cell by breaking down extracellular matrix. Therefore, much attention has been focused on MMP inhibitors to treat cancer. In particular, since synthetic MMP inhibitors have many problems in the selectivity of attack on cancer cells, efforts are being made to find MMP inhibitors from natural products (Kim et al., 2006; Kong et al., 2008; Kim et al., 2018). In this study, we searched for a substance that inhibits adipogenesis and MMPs activity from the halophyte Artemisia scoparia, a native plant growing on the coastal area of South Korea.
As already mentioned earlier, A. scoparia has been used as a folk medicine from ancient times. This plant was collected from the coast of Donggumdo, Ganghwa-gun, Incheon. It was extracted with CH2Cl2 and MeOH. Combined crude extracts were fractionated into n-hexane, 85% aq.MeOH, n-BuOH and water fractions by solvent polarity. The following bioassay methods were used to evaluate the inhibitory effects of A. scoparia on adipogenesis and MMPs
the samples most strongly inhibited formation of lipid droplets in 3T3-L1 adipocytes, and it was almost similar to the control not treated with sample.
In RT-PCR, which measures expression level of obesity-related genes, n-hexane fraction was the most effective at inhibiting mRNA expression of SREBP1C. On the other hand, 85% aq.MeOH fraction was most effective in inhibiting mRNA expression of PPARγ and C/EBPα. In Western blot assay, which measures protein expression level of obesity–related factors, 85%
aq.MeOH fraction was most effective in reducing protein expression levels of all three obesity-related factors SREBP1C, PPARγ and C/EBPα.
In gelatin zymography for measurement of MMP activity, it was confirmed that crude extract significantly reduced enzymatic activity of MMP-9 and MMP-2. Among solvent fractions, n-hexane fraction inhibited enzymatic activity most effectively. In addition, 85% aq.MeOH fraction also showed an enzymatic activity inhibitory effect close to the blank. In ELISA, among the solvent fractions, n-hexane and 85% aq.MeOH fractions showed significant effects on inhibition of MMP-9 and MMP-2 secretion. In case of MMP-2, the secretion was also inhibited by n-BuOH and water fractions. Cell migration related to cancer metastasis and invasion was observed by wound healing assay. The untreated control group showed more migration tendency than the cells treated with the samples. All samples including crude extract were effective in inhibiting cell migration. In RT-PCR, mRNA expressions of MMP-9 and MMP-2 were effectively inhibited by all solvent fractions except water fraction. In Western blot assay, protein expressions of MMP-9 and MMP-2 were most potently inhibited by n-hexane and 85% aq.MeOH fractions. The n-BuOH fraction significantly suppressed protein expressions of MMP-9 and MMP-2.
By bioactivity-guided separation, two compounds were isolated from 85%
aq.MeOH fraction which showed the most potent inhibitory effect both on adipogenesis and MMP enzymatic activity. Their chemical structures were
determined to be known sesquiterpenes, reynosin (1) and santamarine (2) by the extensive 2D NMR experiments. Reynosin and santamarine have been previously isolated from several plants (El-Felary & Chan, 1978; Ito et al., 1984; Adegawa et al., 1987; Fang et al., 2005; Barla et al., 2007; Rosselli et al., 2012; Al-Attas et al., 2015), but were first isolated from the genus artemisia. Reynosin and santamarine have been reported to have anti-inflammatory effects by inhibiting TNF-αproduction in LPS-stimulated RAW 264.7 cells. In addition, these compounds were effective in inhibiting alcohol absorption (Cho et al., 1998; Yoshikawa et al., 2000).
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Appendix
Fig. 18. 1H-1H COSY spectrum of compound 1 isolated from the A. scoparia.
Fig. 20. 1H and 13C NMR spectra of compound 2 isolated from the A. scoparia.
Fig. 21. 1H-1H COSY spectrum of compound 2 isolated from the A. scoparia.
Fig. 22. gHSQC and gHMBC spectra of compound 2 isolated from the A.
scoparia.